The invention relates to new indazole derivatives, processes for their preparation, and their use in medicaments.
EP-A-580 550 describes oxamic acid derivatives which have cholesterol-lowering properties in mammals. The pharmacological characteristic emphasized is the reduction of plasma cholesterol, in particular of LDL cholesterol. Cholesterol-lowering actions are also described in EP-A-188 351 for certain diphenyl ethers having thyroid hormone-like actions, which differ clearly in their chemical structure from the compounds according to the invention.
Indazoles which are connected to a substituted phenyl ring in the 5 position via a bridge member have been disclosed (JP-A-08022109, JP-A-59098060). No thyroid hormone-like properties are described for these 5-substituted indazoles.
An object of the invention is the provision of novel compounds having improved actions, in particular pharmaceutical actions.
It has now been found that compounds of the general formula (I), 
in which
Z represents O, S, CH2, CHF or CF2,
R1 and R2 are identical or different and represent hydrogen, halogen, (C1-C6)-alkyl, CF3, CHF2, CH2, CH2F, vinyl or (C3-C7)-cycloalkyl, where at least one of the two substituents is not equal to hydrogen and is in the ortho position relative to the bridge bond,
R3 represents a head group having an optionally derivatized carboxyl radical, preferably a group of the formula
A-(CH2)nxe2x80x94(CO)mxe2x80x94R8,
in which
A represents CH2, O, S, CO or NR9, in which R9 denotes hydrogen, (C1-C6)-alkyl or (C3-C8)-cycloalkyl or represents the group xe2x80x94(CH2)nxe2x80x94(CO)mxe2x80x94Oxe2x80x94(C1-C4)-alkyl,
n represents the numbers 0 to 3,
m represents the number 0, 1 or 2,
R8 represents hydrogen, hydroxyl, OR10, NR11R12, (C1-C8)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)-aryl, or a saturated, unsaturated or aromatic 5- to 10-membered heterocycle having up to four identical or different heteroatoms from the series S, O and/or N, where the abovementioned radicals are optionally substituted by one, two or three, identical or different substituents from the group halogen, hydroxyl, cyano, nitro, amino, CF3, (C1-C6)-alkyl, (C1-C6)-alkoxy, (C3-C8)-cycloalkyl, xe2x80x94OCOxe2x80x94R13, xe2x80x94COxe2x80x94Oxe2x80x94R14, xe2x80x94COxe2x80x94NR15R16, xe2x80x94NHCOR17 or NHCOOR17, where R10, R11, R12, R13, R14, R15, R16 and R17 are identical or different and in each case represent hydrogen, phenyl, benzyl, (C1-C6)-alkyl, (C1-C6)-alkoxy or (C3-C8)-cycloalkyl, which for their part are optionally mono- or polysubstituted by halogen, hydroxyl, amino, (C1-C4)-alkoxy, xe2x80x94COxe2x80x94O(C1-C4)-alkyl, xe2x80x94NHxe2x80x94COxe2x80x94O(C1-C4)-alkyl, xe2x80x94Oxe2x80x94COxe2x80x94(C1-C4)-alkyl, a heterocycle or optionally halogen- or hydroxyl-substituted phenyl,
R4 and R5 are identical or different and in each case represent hydrogen, hydroxyl, halogen, cyano, nitro, (C1-C4)-alkyl, or the radical of the formula NR18R19, where R18 and R19 have the meaning indicated for R10 and can be identical to or different from this substituent,
R6 represents halogen or has the meaning indicated for R8 and is identical to or different from this substituent or represents the radical 
in which
R20 and R21 together represent oxygen, or are each identical or different and represent hydrogen, halogen, hydroxy, (C1-C6)-alkyl, (C1-C6)-alkoxy or the radical xe2x80x94NR15R16 and
R22 has the meaning of R8 and is identical to or different from this,
R7 represents hydrogen or an acyl group which can be cleaved under physiological conditions with formation of an NH function, preferably hydrogen or acetyl,
and their salts,
preferably the compounds which are mono- or di-ortho-substituted in the phenyl moiety and have a substituent in the 3 position in the indazole ring,
show a pharmacological action and can be used as medicaments or for the production of pharmaceutical formulations.
Heterocycles which may preferably be mentioned in the definition of R8 or of R6 are:
a 5- to 8-membered saturated, partly unsaturated or aromatic optionally benzo-fused heterocycle having up to 4 heteroatoms from the series S, N and/or O, i.e. a heterocycle which can contain one or more double bonds and which is linked via a ring carbon atom or a ring nitrogen atom. Examples which may be mentioned are: tetrahydrofur-2-yl, tetrahydrofur-3-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, pyrrolin-1-yl, piperidin-1-yl, piperidin-3-yl, 1,2-dihydropyridin-1-yl, 1,4-dihydropyridin-1-yl, piperazin-1-yl, morpholin-1-yl, azepin-1-yl, 1,4-diazepin-1-yl, furan-2-yl, furan-3-yl, pyrrol-1-yl, pyrrol-2-yl, pyrrol-3-yl, thienyl, thiazolyl, oxazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyrimidinonyl, pyridazinonyl.
Those preferred from this list are: pyridyl, pyrimidyl, pyridazinyl, pyrimidinonyl, pyridazinonyl and thiophenyl.
Derivatized carboxyl radicals in the definition of the substituent R3 preferably denote groups which can be broken down in the sense of a prodrug to the carboxylic acid or its salts, such as halides, anhydrides, esters or amides.
Alkyl in the context of the invention represents a straight-train or branched alkyl radical preferably having 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 3 carbon atoms. A straight-chain or branched alkyl radical having 1 to 3 carbon atoms is preferred. The following may be mentioned by way of example and preferably: methyl, ethyl, n-propyl, isopropyl, n-, i-, s- or t-butyl, n-pentyl and n-hexyl.
Aryl in the context of the invention represents an aromatic radical preferably having 6 to 10 carbon atoms. Preferred aryl radicals are phenyl and naphthyl.
Cycloalkyl in the context of the invention represents a cycloalkyl group preferably having 3 to 8, 3 to 7 or 3 to 6 carbon atoms. The following may be mentioned by way of example and preferably: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
Alkoxy in the context of the invention preferably represents a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms. A straight-chain or branched alkoxy radical having 1 to 3 carbon atoms is preferred. The following may be mentioned by way of example and preferably: methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-pentoxy and n-hexoxy.
Halogen in the context of the invention includes fluorine, chlorine, bromine and iodine. Fluorine, chlorine or bromine is preferred.
Depending on the substitution pattern, the compounds according to the invention can exist in stereoisomeric forms which either behave as image and mirror image (enantiomers), or which do not behave as image and mirror image (diastereomers). The invention relates both to the enantiomers or diastereomers and their respective mixtures. Like the diastereomers, the racemic forms can be converted into the stereoisomerically uniform constituents in a known manner.
Certain compounds can furthermore be present in tautomeric forms. This is known to the person skilled in the art, and compounds of this type are likewise encompassed by the scope of the invention.
The compounds according to the invention can also be present as salts. Physiologically acceptable salts are preferred in the context of the invention.
Physiologically acceptable salts can be salts of the compounds according to the invention with inorganic or organic acids. Preferred salts are those with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulphuric acid, or salts with organic carboxylic or sulphonic acids such as acetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid or naphthalenedisulphonic acid.
Physiologically acceptable salts can likewise be salts of the compounds according to the invention with bases, such as metal or ammonium salts. Preferred examples are alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. magnesium or calcium salts), and also ammonium salts which are derived from ammonia or organic amines, such as ethylamine, di- or triethylamine, ethyldiisopropylamine, monoethanolamine, di- or tri-ethanolamine, dicyclohexylamine, dimethylaminoethanol, dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine, methylpiperidine, arginine, lysine, ethylenediamine or 2-phenylethylamine.
The compounds according to the invention can also be present in the form of their solvates, in particular in the form of their hydrates.
Preferred compounds of the general formula (I) are those
in which
Z represents O, CH2 or CF2,
R1 and R2 are identical or different and represent hydrogen, fluorine, chlorine, bromine, (C1-C4)-alkyl, CF3, CHF2, CH2F, vinyl or (C3-C6)-cycloalkyl, where at least one of the two substituents is not equal to hydrogen and is in the ortho position relative to the bridge bond, in particular both substituents are not equal to hydrogen and both are in the ortho position,
R3 represents a head group having an optionally derivatized carboxyl radical, preferably a group of the formula
A-(CH2)nxe2x80x94(CO)mxe2x80x94R8,
which is preferably in the para position relative to the bridge bond,
in which
A represents CH2, O or NR9, in which R9 denotes hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl or represents the group xe2x80x94(CH2)nxe2x80x94(CO)mxe2x80x94Oxe2x80x94(C1-C4)-alkyl,
n represents the number 0 or 1,
m represents the number 1 or 2,
R8 represents hydrogen, hydroxyl, OR10, NR11R12, (C1-C8)-alkyl (C3-C8)-cycloalkyl, (C6-C10)-aryl, or a saturated, unsaturated or aromatic 5- to 10-membered heterocycle having up to four identical or different heteroatoms from the series S, O and/or N, where the abovementioned hydrocarbon radicals and heterocycles are optionally substituted by one, two or three identical or different substituents from the group halogen, hydroxyl, cyano, nitro, amino, CF3, (C1-C4)-alkyl, (C1-C4)-alkoxy, (C1-C4)-alkoxyphenyl, (C3-C8)-cycloalkyl, xe2x80x94Oxe2x80x94COxe2x80x94R13, xe2x80x94COxe2x80x94Oxe2x80x94R14, xe2x80x94COxe2x80x94NR15R16, xe2x80x94NHCOR17 or xe2x80x94NHCOOR17, where R10, R11, R12, R13, R14, R15, R16 and R17 are identical or different and each represent hydrogen, benzyl, (C1-C6)-alkyl or (C3-C8)-cycloalkyl, which for their part are optionally mono- or polysubstituted by fluorine, chlorine, hydroxyl, amino, xe2x80x94COxe2x80x94Oxe2x80x94(C1-C4)-alkyl, xe2x80x94NHxe2x80x94COxe2x80x94O(C1-C4)-alkyl, xe2x80x94Oxe2x80x94COxe2x80x94(C1-C4)-alkyl, imidazolyl, hydroxyphenyl or (C1-C4)-alkoxy,
R4 and R5 are identical or different and each represent hydrogen, halogen or (C1-C4)-alkyl,
R6 represents chlorine, fluorine, bromine or has the meaning indicated for R8 and is identical to or different from this abovementioned substituent or represents the radical 
in which
R20 and R21 together represent oxygen, or are each identical or different and represent hydrogen, halogen, hydroxyl, (C1-C4)-alkyl, (C1-C4)-alkoxy or the radical xe2x80x94NR15R16 and
R22 has the meaning of R8 and is identical to or different from this,
R7 represents hydrogen,
and their salts.
Compounds of the general formula (I) of particular importance are those in which
Z represents CH2 or in particular oxygen,
R1 and R2 are identical or different and represent methyl, ethyl, propyl, isopropyl, chlorine, bromine, CF3, vinyl or cyclopropyl, where both substituents are in the ortho position to the bridge bond,
R3 represents the group xe2x80x94Oxe2x80x94(CH2)nxe2x80x94COxe2x80x94R8 or preferably the group NR9xe2x80x94(CH2)nxe2x80x94(CO)mxe2x80x94R8, which is each in the para position relative to the bridge bond, where
R9 denotes xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94(C1-C4)-alkyl, substituted (C1-C4)-alkyl, (C3-C7)-cycloalkyl, xe2x80x94COxe2x80x94COxe2x80x94Oxe2x80x94(C1-C4)-alkyl or in particular hydrogen,
m represents the number 1 or in particular 2,
n represents the number 1 or 0,
R8 represents methyl, ethyl, n-propyl, isopropyl, n-, i-, s- or t-butyl, n-pentyl or n-hexyl, thiophenyl, pyridyl, or the groups xe2x80x94CH2xe2x80x94O-benzyl, OR10 or NR11R12,
where R10 represents hydrogen, or optionally hydroxyl-substituted straight-chain or branched alkyl having up to 7 carbon atoms,
where R11 and R12 are identical or different and represent hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-pentoxy, n-hexoxy or straight-chain or branched alkyl having up to 6 carbon atoms, where alkyl for its part is optionally mono- or polysubstituted identically or differently by hydroxyl, xe2x80x94COxe2x80x94O(C1-C4)-alkyl, xe2x80x94NHxe2x80x94COxe2x80x94O(C1-C4)-alkyl, imidazolyl and/or hydroxyphenyl,
R4 and R5 represent methyl, fluorine or chlorine or in particular hydrogen,
R6 represents hydrogen, hydroxyl, OR10, NR11R12, methyl, ethyl, n-propyl, isopropyl n-, i-, s- or t-butyl, n-pentyl, (C3-C6)-cycloalkyl, (C6-C10)-aryl, or represents a saturated, unsaturated or aromatic 5- to 10-membered heterocycle having up to three identical or different heteroatoms from the series S, O and/or N, where the abovementioned hydrocarbon radicals and heterocycles are optionally substituted by one, two or three identical or different substituents from the group halogen, hydroxyl, cyano, nitro, amino, CF3, (C1-C4)-alkyl, (C1-C4)-alkoxy, (C3-C8)-cycloalkyl, Oxe2x80x94COxe2x80x94R13, xe2x80x94COxe2x80x94Oxe2x80x94R14, xe2x80x94COxe2x80x94NR15R16 or xe2x80x94NHCOOR17, where R10, R11, R12, R13, R14, R15, R16 and R17 are identical or different and each represent hydrogen, benzyl, (C1-C4)-alkyl or (C3-C6)-cycloalkyl, which for their part are optionally substituted by amino or (C1-C4)-alkoxy,
or represents the radical 
in which
R20 and R21 together represent oxygen, or are each identical or different and represent hydrogen, halogen, hydroxyl, (C1-C4)-alkyl, (C1-C4)-alkoxy or the radical xe2x80x94NR15R16 and
R22 represents hydrogen, hydroxyl, OR10, NR11R12, (C1-C4)-alkyl, (C3-C6)-cycloalkyl, (C6-C10)-aryl, or a saturated, unsaturated or aromatic 5- to 10-membered heterocycle having up to three identical or different heteroatoms from the series S, O and/or N, where the abovementioned hydrocarbon radicals and heterocycles are optionally substituted by one, two or three identical or different substituents from the group halogen, hydroxyl, cyano, nitro, amino, CF3, (C1-C4)-alkyl, (C1-C4)-alkoxy, (C3-C8)-cycloalkyl, Oxe2x80x94COxe2x80x94R13, xe2x80x94COxe2x80x94Oxe2x80x94R14, xe2x80x94COxe2x80x94NR15R16 or xe2x80x94NHCOOR17, where R10, R11, R12, R13, R14, R15, R16 and R17 are identical or different and each represent hydrogen, benzyl, (C1-C4)-alkyl or (C3-C6)-cycloalkyl, which for their part are optionally substituted by amino or (C1-C4)-alkoxy,
R7 represents hydrogen,
and their salts.
Particularly preferred compounds of the formula (I) are those in which Z is oxygen.
Particularly preferred compounds of the formula (I) are those in which R3 is the group xe2x80x94NHxe2x80x94COxe2x80x94COxe2x80x94R8 or xe2x80x94Oxe2x80x94CH2xe2x80x94COxe2x80x94R8 and R8 is a group which in the sense of a prodrug can be broken down to the carboxylic acid or its salts. Particularly preferred compounds of the formula (I) are those in which R4, R5 and R7 represent hydrogen.
Particularly preferred compounds of the formula (I) are those in which R1 and R2 are both located in the ortho position relative to Z and represent bromine, trifluoromethyl, cyclopropyl and in particular methyl.
The compounds of the general formula (I) according to the invention can be prepared by reacting reactive indazole derivatives of the general formula (II) with reactive phenyl derivatives of the general formula (III) 
where the substituents R1, R2, R4, R5, R6 and R7 have the meaning indicated above and
R3xe2x80x2 has the meaning indicated for R3 or represents NO2 or NPG,
where PG is a protective group,
X and Y each represent groups of opposite reactivity, where, for example, X can be an electrophilic radical which reacts with a nucleophilic Y substituent and vice versa,
Zxe2x80x2 has the meaning indicated for Z or represents 
if appropriate in the presence of inert solvents and catalysts and if appropriate with isolation of the intermediates of the general formula (IV), or directly to give compounds of the formula (I).
Catalysts which may be mentioned by way of example are coupling catalysts such as Pd and/or Cu compounds.
The following may be mentioned by way of example for the reactive groups X and Y:halogen, hydroxyl, CH2Br, mercapto, CHO, Li, MgHal, Sn or boron derivatives.
The indazoles of the general formula (II) employable according to the invention are known or can be prepared according to known methods (compare, for example, Ainsworth et al., J. Amer. Chem. Soc., 80, 1958, 965; Ainsworth et al., J. Amer. Chem. Soc., 79, 1957, 5245; Piozzi, F. et al., Gazz. Chim. Ital., 93 1963, 3-14; Simon, U, et al., Justus Liebigs Ann. Chem. 697, 1966, 17-41).
The phenyl derivatives of the general formula (III) are likewise known or can be prepared according to known methods (compare, for example, EP 580 550 A).
The reaction of the starting compounds (II) with (III) in general proceeds at normal pressure. However, it can also be carried out at elevated or reduced pressure.
The reaction can be carried out in a temperature range from xe2x88x92100xc2x0 C. to 200xc2x0 C., preferably between from xe2x88x9278xc2x0 C. and 150xc2x0 C., in the presence of inert solvents. Inert solvents which may preferably be mentioned are: dimethyl sulphoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), diethyl ether etc.
Depending on the specific substituent pattern, in the reaction of (II) and (III) intermediates of the formula (IV) or (IVxe2x80x2) can also result, in which, for example, the substituent R3 represents a nitro group or Z represents a CHOH group, which can then be reduced to the corresponding amino groups or methylene groups according to customary methods with or without isolation of these intermediates and subsequently reacted further according to customary methods with carboxylic acids or carboxylic acid derivatives such as esters, anhydrides or halides to give amide compounds of the formula (I).
The process according to the invention can be illustrated by way of example by the following reaction schemes: 
Depending on the meaning of the substituents R1, R2, R4, R5, R6, R7, it may be useful or necessary to vary these in individual process steps within the scope of meaning indicated.
The precursors and intermediates of the formula (IV) occurring in the reaction of (II) and (III) which are marked in the above reaction scheme by (IVxe2x80x2) and (IVxe2x80x3) are new. This application therefore also relates to compounds of the general formula (IV) 
in which
R1, R2, R4, R5, R6 and R7 have the meaning indicated above for formula (I),
Zxe2x80x2 has the meaning indicated for Z or represents CHOH, and
R3xe2x80x2 has the meaning indicated for R3 or represents NO2 or NPG, where PG represents a protective group.
Protective groups (PG) are understood in the present application as meaning those groups in starting materials, intermediates and/or final products which protect functional groups present, such as carboxyl, amino or hydroxyl groups, and which are customary in preparative organic chemistry. The groups protected in this way can then be converted into free functional groups under known conditions in a simple manner.
The compounds of the formula (I) according to the invention show a surprising and valuable pharmacological spectrum of action and can therefore be employed as versatile medicaments. In particular, they can be employed in all indications which can be treated using natural thyroid hormones, such as, by way of example and preferably, depression, goitre or thyroid cancer. Arteriosclerosis, hypercholesterolaemia and dyslipidaemia can preferably be treated using the compounds of the formula (I) according to the invention. Adiposity and obesity and cardiac insufficiency can moreover also be treated and a postprandial lowering of the triglycerides achieved.
The compounds are also suitable for the treatment of certain respiratory tract disorders, namely in particular of pulmonary emphysema and for medicinally promoting maturation of the lungs.
The compounds are furthermore suitable for the treatment of pain and migraine, for neuronal repair (remyelinization) and for the treatment of Alzheimer""s disease.
The compounds are furthermore suitable for the treatment of osteoporosis, cardiac arrhythmias, hypothyroidism and skin disorders.
Moreover, the compounds can also be employed for promotion and regeneration of hair growth and for the treatment of diabetes.
The active compounds according to the invention open up a further treatment alternative and are an enrichment of pharmacy. In comparison with the known and previously employed thyroid hormone preparations, the compounds according to the invention show an improved spectrum of action. They are preferably distinguished by great specificity, good tolerability and lower side effects, in particular in the cardiovascular area.
The efficacy of the compounds according to the invention can be tested, for example, in vitro by the T3 promoter assay cell test described below:
The test is carried out using a stably transfected, human HepG2 hepatocarcinoma cell which expresses a luciferase gene under the control of a thyroid hormone-regulated promoter. The vector used for the transfection carries a minimal thymidine kinase promoter ahead of the luciferase gene having a thyroid hormone-responsive element (TRE) which consists of two inverted palindromes of 12 bp each and one 8 bp spacer.
For the test, the cell cultures are inoculated into 96 well plates in Eagle""s Minimal Essential Medium with the following additives: glutamine, tricine [N-(tris-(hydroxymethyl)-methyl)-glycine], sodium pyruvate, non-essential amino acids (L-Ala, L-Asn, L-Asp, L-Pro, L-Ser, L-Glu, Gly), insulin, selenium and transferrin. The cultures are grown at 37xc2x0 C. and under a 10% CO2 atmosphere for 48 hours. Serial dilutions of test substance or reference compound (T3, T4) and costimulator retinolic acid are then added to the test cultures and these are incubated for a further 48 or 72 hours as beforehand. Each substance concentration is tested in four replicates. For the determination of the luciferase induced by T3 or other substances, the cells are then lysed by addition of a Triton- and luciferase-containing buffer (Promega) and immediately measured luminometrically. The EC50 values of each compound are calculated. Representative results for the compounds according to the invention are shown in Table 1:
The compounds according to the invention also surprisingly show advantageous properties in the tests described below:
Description of tests for finding pharmacologically active substances:
The substances which are to be investigated in vivo for their serum cholesterol-lowering action are administered orally to male mice having a body weight of between 25 and 35 g. The animals are divided into groups having an equal number of animals, as a rule n=7-10, one day before the start of the experiment. During the entire experiment, drinking water and feed are available to the animals ad libitum.
The substances are administered orally once daily for 7 days. For this purpose, the test substances are dissolved, for example, in a solution of Solutol HS 15+ethanol+saline solution (0.9%) in the ratio 1+1+8 or in a solution of Solutol HS 15+saline solution (0.9%) in the ratio 2+8. The dissolved substances are administered in a volume of 10 ml/kg of body weight using a stomach tube. The control group used is animals which have been treated exactly the same, but contain only the solvent (10 ml/kg of body weight) without test substance.
Before the first administration of substance, blood is taken from each mouse by puncture of the retroorbital venous plexus for the determination of the serum cholesterol (preliminary value). The test substance is then administered to the animals for the first time using a stomach tube. 24 hours after the last administration of substance (on the 8th day after the start of treatment), blood is again taken from each animal by puncture of the retroorbital venous plexus to determine the serum cholesterol. The blood samples are centrifuged and, after recovering the serum, the cholesterol is determined photometrically using an EPOS analyser 5050 (Eppendorf-Gerxc3xa4tebau, Netheler and Hinz GmbH, Hamburg). The determination is carried out using a commercially available enzyme test (Boehringer Mannheim, Mannheim).
The action of the test substances on the serum cholesterol concentration is determined by subtraction of the cholesterol value of the 1st blood sample (preliminary value) from the cholesterol value of the 2nd blood sample (after treatment). The differences of all cholesterol values of a group are averaged and compared with the average value of the differences of the control group.
Statistical analysis is carried out using Student""s t test after prior testing of the variances for homogeneity.
Substances which statistically significantly (p less than 0.05) lower the serum cholesterol of the treated animals by at least 10% compared with that of the control group are regarded as pharmacologically active.
At the end of the experiment, the animals are weighed and sacrificed after taking blood. To check for potential cardiovascular side effects under the action of substance, the hearts are removed and weighed. An effect on the cardiovascular system can be determined by means of a significant increase in the heart weight. A further parameter which can be used for the substance action is a change in body weight.
In an analogous manner, for example, NMRI mice, ob,ob mice, Wistar rats or fa,fa diabetic rats can be used as experimental animals for this test.
A further in vivo test in which the compounds according to the invention surprisingly show advantageous properties is the cholesterol-fed rat animal model [A. Taylor et al., Molecular Pharmacology 52, 542-547 (1997); Z. Stephan et al., Atherosclerosis 126, 53-63 (1996)].
Furthermore, the cholesterol-lowering action of the compounds according to the invention can also be tested on normocholesterolaemic dogs by oral administration of the test substances for 5 to 7 days.
For the further investigation of potential cardiovascular side effects under the action of substance, inter alia the determination of the expression of the mRNA of the xe2x80x9cHCN2xe2x80x9d ion channel (xe2x80x9chyperpolarization-activated cyclic nucleotide-gated channelxe2x80x9d) in mouse or rat hearts can be used [cf. also: Trost et al., Endocrinology 141 (9), 3057-3064 (2000); Gloss et al., Endocrinology 142 (2), 544-550 (2001); Pachuki et al., Circulation Research 85, 498-503 (1999)]:
HCN2 Assay:
The quantification of the mRNa of the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN2) in rat hearts was carried out by means of real-time PCR (TaqMan PCR; Heid et al., Genome Res. 6 (10), 986-994). To this end, after preparation of the hearts the total RNA is isolated by means of RNaesy columns (Qiagen), digested with DNase and then transcribed into cDNA (SUPERSCRIPT II RT cDNA synthesis kit, from Gibco). The HCN2 mRNA determination is carried out on an ABI Prism 7700 apparatus (from Applied Biosystems). The sequence of the forward and reverse primers read: 5xe2x80x2-GGGAATCGACTCCGAGGTC-3xe2x80x2 or 5xe2x80x2-GATCTTGGTGAAACGCACGA-3xe2x80x2, that of the fluorescent sample 5xe2x80x2-6FAM-ACAAGACGGCCCGTGCACTACGC-TAMRA-3 (FAM=fluorescent dye 6-carboxyfluorescein; TAMRA=quencher 6-carboxytetramethylrhodamine). During the polymerase chain reaction, the fluorescent dye FAM is removed by means of the 5xe2x80x2-exonuclease activity of the taq polymerase and the previously quenched fluorescence signal is thereby obtained. The xe2x80x9cthreshold cyclexe2x80x9d (Ct value) recorded is the number of cycles at which the fluorescence intensity was 10 standard deviations above the background fluorescence. The relative expression of the HCN2 mRNA calculated hereby is then standardized to the expression of the ribosomal protein L32.
In an analogous manner, this assay can also be carried out using mice hearts. The sequence of the forward and reverse primer in this case read 5xe2x80x2-CGAGGTGCTGGAGGAATACC-3xe2x80x2 or 5xe2x80x2-CTAGCCGGTCAATAGCCACAG-3xe2x80x2, that of the fluorescent sample 5xe2x80x2-6FAM-CATGATGCGGCGTGCCTTTGAG-TAMRA-3.
All customary administration forms are suitable for the administration of the compounds of the general formula (I), i.e. oral, parenteral, inhalant, nasal, sublingual, buccal, rectal or external, e.g. transdermal, in particular preferably oral or parenteral. In the case of parenteral administration, intravenous, intramuscular or subcutaneous administration may be mentioned in particular, e.g. as a subcutaneous depot. Oral administration is very particularly preferred.
In this case, the active compounds can be administered on their own or in the form of preparations. Suitable preparations for oral administration are, inter alia, tablets, capsules, pellets, coated tablets, pills, granules, solid and liquid aerosols, syrups, emulsions, suspensions and solutions. In this case, the active compound must be present in such an amount that a therapeutic action is achieved. In general, the active compound can be present in a concentration of 0.1 to 100% by weight, in particular 0.5 to 90% by weight, preferably 5 to 80% by weight. In particular, the concentration of the active compound should be 0.5 to 90% by weight, i.e. the active compound should be present in amounts which are sufficient to achieve the dosage range indicated.
For this purpose, the active compounds can be converted into the customary preparations in a known manner. This is carried out using inert, non-toxic, pharmaceutically suitable carriers, excipients, solvents, vehicles, emulsifiers and/or dispersants.
Excipients which may be mentioned by way of example are: water, non-toxic organic solvents such as paraffins, vegetable oils (e.g. sesame oil), alcohols (e.g. ethanol, glycerol), glycols (e.g. polyethylene glycol), solid carriers such as natural or synthetic ground minerals (e.g. talc or silicates), sugars (e.g. lactose), emulsifiers, dispersants (e.g. polyvinylpyrrolidone) and lubricants (e.g. magnesium sulphate).
In the case of oral administration, tablets, of course, can also contain additives such as sodium citrate together with additional substances such as starch, gelatin and the like. Aqueous preparations for oral administration can furthermore be mixed with flavour enhancers or colourants.
In the case of oral administration, doses of 0.001 to 5 mg/kg, preferably 0.005 to 3 mg/kg, of body weight are preferably administered every 24 hours.
The new active compounds can be administered on their own and, if required, also in combination with other active compounds, preferably from the group consisting of CETP inhibitors, antidiabetics, antioxidants, cytostatics, calcium antagonists, blood pressure-lowering agents, thyroid hormones, inhibitors of HMG-CoA reductase, inhibitors of HMG-CoA reductase gene expression, squalene synthesis inhibitors, ACAT inhibitors, circulation-promoting agents, platelet aggregation inhibitors, anticoagulants, angiotensin II receptor antagonists, cholesterol absorption inhibitors, MTP inhibitors, aldose reductase inhibitors, fibrates, niacin and PPAR agonists.